1. TITLE OF INVENTION
Soluble leishmania antigen specific whole blood assay as a marker of asymptomatic
infection and disease status in human Visceral leishmaniasis
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2. FIELD OF INVENTION
The present invention provides soluble leishmania antigen specific detection of IFN-y
and IL-10 in whole blood of cured VL and active VL patients respectively for the purpose of
detection of asymptomatic infection and disease status in human visceral leishmaniasis. The
invention contribute to the development of better markers for infection and to understanding
T cell immune response in the high endemic foci of VL. Further, the invention is crucial for
intervention studies on vaccine or vector control and for monitoring of ongoing transmission
in endemic area.
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3.Backsround of invention with resard to drawback associated with
prior art:
Visceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease caused
by obligate intracellular parasites of the genus Leishmania and is a serious public health
problem in several endemic tropical and subtropical regions. The estimated annual global
incidence of VL is 500,000 and 90 % of these cases occur in India, Nepal, Bangladesh,
Sudan and Brazil. In 2005, the governments of India, Bangladesh and Nepal signed a
memorandum of understanding for VL elimination from the Indian subcontinent by the year
2015 with a target to reduce the incidence of VL disease below 1 per 10,000 per year. One of
the challenges faced by this elimination initiative is that only a small proportion of
L.donovani infection manifests as clinical disease, and some authors suggest that the latent
carriers may constitute a reservoir of parasites driving the epidemic. In Bihar, India, the ratio
of new clinical cases to asymptomatic individuals was estimated as one to 10 -II, while in
Bangladesh it was I to 4. However, neither the role of these asymptomatic carriers in
transmission nor the prognosis of asymptomatic infection at the individual level is fully
elucidated. Research in this area is compounded by the absence of validated markers for
L.donovani infection, as so far diagnostic tests for VL have been evaluated mostly on their
capacity to detect clinical disease.
Various tests to detect parasite DNA, like PCR and qPCR, have been recently described but
the sensitivity of such methods seems to be variable, depending on the choice of target
sequences and due to the short half life of DNA in the body (24 hrs). Serological tests such as
the Direct Agglutination Test (DAT) and rk39 ELISA have been used for epidemiological
work but these tests are poorly characterized for the purpose of detecting asymptomatic
L.donovani infection. Antibody response may be low relative to the cellular immune
response in exposed, asymptomatic individuals. The first described and most straightforward
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test to detect cellular immunity is the Leishmanin Skin Test (LSI), a measure of the delayedtype
hypersensitivity reaction. However, its complex administration requiring reading 48
hours after intra-dermal injection, the current scarcity of GMP-grade leishmanin antigen and
its low sensitivity when evaluated on Indian patients are major drawbacks. Hence more
appropriate tools for the evaluation of the cellular response to Leishmania infection in the
field are urgently needed. The QuantiFERON tube test (Cellestis, Australia) is a commercial
test kit assessing the cellular immune response in Mycobacterium tuberculosis infections. It
measures IFN-y levels released by sensitized T-lymphocytes in a venous blood sample
collected in special tubes after stimulation with peptide antigens representing two M
tuberculosis proteins. We recently developed a modified version of this IFN-yRA assay to
detect sub-clinical L.donovani infection and reported promising results with a system
employing Soluble Leishmania Antigen (SLA). In addition to detecting IFN-y secretion by
whole blood cells from roughly one quarter of healthy individuals living in the VL endemic
zone, the assay surprisingly revealed IFN-y secretion by cells from the majority of patients
with active VL. This is in sharp contrast to the experience involving PBMCs in which
antigen-specific IFN-y production is typically absent. We have also recently reported IL-10
secretion by whole blood cells from VL patients, suggesting that the profile of multiple
cytokine release might better reflect disease status than IFN-y production alone. In the
current studies, we have evaluated the ability of the assay to detect antigen-specific release of
IFN-y and IL-10 by whole blood cells obtained from a large series of clinically well
characterized subjects, including confirmed VL cases with active disease, clinically cured VL
cases, and healthy individuals from endemic and non-endemic areas. The findings strongly
reinforce the utility of the whole blood assay for the detection of IFN-y production by L.
donovani infected, asymptomatic individuals, and for detection of IL-10 secretion as the
signature cytokine distinguishing active VL from cured or asymptomatic cases.
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4, Objective of invention
The main objective of this invention is to develop the whole blood assay with SLA as
a marker of infection and disease status in human visceral leishmaniasis which is
crucial for intervention studies on vaccine or vector control for monitoring of ongoing
transmission in endemic area.
5. Statement of invention
The present invention provides Soluble leishmania antigen specific detection of
IFN-y and lL-10 in whole blood of cured VL and activel VL patients respectively for
the purpose of detection of asymptomatic infection and disease status in human
visceral leishmaniasis. The whole blood assay with SLA showed a potential as marker
of infection and is crucial for intervention studies on vaccine or vector control for
monitoring of ongoing transmission.
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6. A summary of invention
In endemic area, a large number of exposed individual are able to mount a protective cellular
immune response against Leishmania and either eliminate infection or remain asymptomatic
carriers. Leishmanin skin test (LST) or serological methods have been used to document
incident infection, but there value as marker has not been firmly established so far. Here we
tested the Quantiferon assay to document the leishmanial infection. We employed the whole
blood assay to evaluate IFN-y and lL-10 production in 35 patients with active visceral
leishmaniasis (VL), 54 cured VL, 27 patients with other diseases and 52 Non-Endemic
Healthy Controls (NEHC). We also tested the assay on 147 Endemic Healthy Controls
(EHC), all close contacts of VL cases having a high probability of Z. donovani infection, and
correlated their cellular response with their serological antibody titers against L. donovani
and Phlebotomus argentipes saliva. The whole blood assay detected IFN-y release in 24% of
the EHCs, and these individuals also had elevated titers of anti-saliva antibodies, consistent
with their having had a higher risk of exposure to an infected sand fly bite. The whole blood
cells from the majority of both active (80%) and cured (85%) VL patients showed
significantly elevated levels of antigen specific IFN-y production. Only the active VL patients
produced IL-10, which can therefore be assayed in conjunction with IFN-y to distinguish
active cases from clinically exposed, immune individuals.
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7. A brief description of the accompanying drawing
Table 1:
Positive and negative agreement indices (Al) between Cellular immunity (QuantiFERON i.e
Whole blood IFN-y release assay) and serology (DAT and rk39 ELISA) in Endemic Healthy
Subjects (n= 147).
Figure 1: : IFN-y levels in plasma after stimulation of whole blood cells with PBS (Nil),
SLA or PHA. (a) Non endemic healthy controls (NEHC, n= 52), (b) Cured VL, 6 month after
treatment (n=54), (c) Endemic healthy controls (EHC, n=147), (d) Active VL (n=35), (e)
Comparison of baseline concentration (Nil value) of IFN-y (lU/ml) in different subject
groups, (f) Comparison of SLA stimulated IFN- y release (NIL value subtracted) for
different subject groups. Bars represent the mean values (a-d) or median values (e-f) and the
dotted line indicates the cutoff (0.78IU/ml) for positivity by ROC and Figure If compares
the results in each study group to this cut off value. The assay was positive in 46/54 (85.2%)
of subjects with cured VL while none of the NEHC was positive. Therefore, the IFN-yRA
had a sensitivity of 85.2% (95% CI 73.4 - 92.3) in cured VL patients to detect the cellular
immune response and a specificity of 100% (95% CI 93.1 - 100.0) in NEHC at the optimal
cut off. Asterisks indicate statistical significance between the indicated groups ( ns= not
significant, * p <0.05 , ** P< 0.01 , *** p <0.001 )
Figure 2. Antigen specific IFN-y response in whole blood of active VL patients (n=l8) after
plasma replacement using pooled plasma from non-endemic healthy individuals to exclude
the possible stimulatory effect of anti-leishmanial antibodies forming immune-complexes
with the SLA, there was still increased antigen-specific IFN- y production in 17 of 18
samples.
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Figure 3. Human IgG response to Phlebotomous argentipes saliva. Comparison of NEHC
(n= 13), EHCs negative for IFN-yRA (n=21), EHCs positive for IFN-yRA (n=35) and
Active VL (n=24). Sera were processed for antibodies to P.argentipes saliva by a modified
ELISA with a P. papatasi saliva pre-adsorption step. This analysis revealed a significantly
higher level of antibodies in the IFN-yRA positive EHCs compared to the EHCs who were
IFN-yRA negative or to the NEHCs, and comparable to the elevated anti-saliva titers
observed in the active VL cases. To the extent that these elevated titers reflect a greater
exposure to sand fly bites and thus a greater risk of exposure to infected flies, then the
findings support the conclusion that the Whole blood IFN-y release assay detects sub-clinical
infections in the EHCs.
Figure 4. lL-10 levels in plasma after stimulation of whole blood cells with PBS (Nil), SLA
or PHA. (a) NEHC (n= 33), (b) Cured VL, 6 month after treatment (n=38), (c) EHC (n=64),
(d) Active VL (n=35), (e) Comparison of baseline concentration of lL-10 (pg/ml) in different
subject groups (Nil value ), (f) Comparison of lL-10 levels against SLA (NIL value
subtrated) for different subject groups. Bars represent the mean values (a-d) or median values
(e-f). Asterisks indicate statistical significance between, the groups indicated ( ns= not
significant, * p <0.05 , ** P< 0.01 , *** p <0.001 ). The plasma IL-10 levels from active VL
cases were elevated even in the non-stimulated cultures (Fig 4e), consistent with the elevated
circulating levels of IL-10 previously described in patients with active disease. The likelihood
that at least some of the circulating IL-10 is produced by antigen specific cells present in the
peripheral blood is supported by the current results in which the whole blood cells from 30 of
33 active VL cases were driven by SLA to secrete higher levels of ILIO (Fig 4f). Thus
antigen-specific IL-10 secretion appears to be a marker of disease status in human VL, and in
conjunction IFN-yRA , can be used to distinguish active cases from clinically exposed,
immune individuals.
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8. Detailed Discriytion of Invention
The study was approved by the Ethics Committee of the Banaras Hindu University,
Varanasi. Written informed consent was obtained from all of the subjects included in the
study, who were divided into 4 clinically well characterized groups: i) 35 cases of
parasitologically confirmed, active VL; ii) 54 subjects who were definitively cured of VL
and sampled at least 6 months after successful treatment when they would be expected to
have reconstituted a strong cellular immune response; iii) 27 subjects suffering from other
febrile illnesses like malaria, tuberculosis, typhoid fever, dengue that could potentially lead to
cross-reaction in the IFN-yRA; iv) 52 healthy controls living in non-endemic regions (NEHC)
who are not expected to react in the IFN-yRA. The cured VL patients and the NEHC groups
were used as the denominators to estimate sensitivity and specificity, respectively, of the
IFN-y RA, see below. Furthermore, we compared the performance of the modified
QuantiFERON assay with that of serology in a series of 147 endemic healthy controls (EHC)
who were close contacts of VL cases recruited at community level in Bihar, i.e. a group with
a high probability of being asymptomatically infected by L.donovani. We selected 104
subjects from 88 households with at least one confirmed VL case in the past 5 years, as well
as 43 neighbors of such cases. We excluded subjects who suffered from kala-azar in the past,
those having fever within the past month, and children less than five years of age. All
subjects within the EHC group were followed up 6 and 12 months after enrolment to monitor
any development of active VL.
Whole Blood Assay :
The QuantiFERON (Cellestis, Australia) based whole blood assay was conducted
according to the manufacturer's instructions and to modifications described previously.
Briefly, 1 ml of venous blood was collected in QuantiFERON assay kit tubes (1 for SLA
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stimulation, 1 for a positive PHA and 1 for a negative control PBS) and hand shaken to mix
with heparin. SLA at a concentration of 5 fig/ml was added to the test tube within 2 hrs to a
37 C incubation for 16- 24 hrs. Incubated samples were centrifuged at 2000 x g for 10 min
and harvested plasma stored at -20°C until used for IFN-y analysis. SLA was prepared from
stationary-phase promastigotes of Z. donovani strain as described previously. Briefly 2x 10^
metacyclic promastigote were harvested and centrifuged at 4500 rpm for 20 minutes to obtain
the pellet. Pellet so obtained was washed thrice with cold PBS and then resuspended to
lOmM TRIS-HCI, 1 mM EDTA (pH8.0) 1.6 mM PMSF and 50 ng/ml leupeptin to obtain a
concentration of 2x10^ parasites/ml. Now it was sonicated 4-5 times for 15 seconds at 10 Hz
and centrifuged at 27000xg for 30 minutes at 4°C. After removing the lipid layer from the
surface of supernatant, remaining solution were ultracentrifliged at lOOOOOxg for 4 hrs at 4°C.
The supernatant so obtained was stored at -80°C until use. Protein was estimated using BCA
method (Pierce). PHA and phosphate-buffered saline (PBS) were used as positive and
negative controls, respectively.
IFN-y levels in plasma samples from stimulated blood samples was measured using
the QuantiFERONELlSA kit (provided by Cellestis, Australia) and as per manufacturer's
instructions. A 4-point standard curve of a known amount of IFN-y in International Unit per
ml (lU/ml) was used to determine the level of IFN-y produced in response to SLA
stimulation. IFN-y levels (expressed in lU/mL) of each test sample were determined by
subtracting background levels measured in the corresponding non-stimulated (PBS) sample.
If the positive control tube with mitogen (PHA) did not show stimulation, the test result was
to be disqualified. IL-IO level in plasma samples from stimulated blood samples were
measured using matched antibody pairs (BD Pharmingen) by ELISA and its concentrations
(expressed in pg/ml) were calculated using a standard curve generated from recombinant
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cytokines. Antibody response against Phlebotomus argentipes saliva was measured by
ELISA using preabsorbtion methods as described Gidwani et. al.
ELISA using recombinant antigen rK39:
Microtiter plates (Maxisorb, Nunc,, Denmark) were coated with 25ng recombinant
rK39 antigens in 0.0 IM carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. Next day,
wells were washed with PBS containing 0.05% Tween-20 and blocked with 1% bovine serum
albumin (BSA) in PBS Tween for 2 hrs. at 25°C. After washing, lOOfxl of human sera diluted
1:400 in PBS Tween were incubated for 1 hr at 25°C, followed by 100)al of HRP conjugated
anti-human IgG (Invitrogen) diluted 1:32000 for 30 min. at 25°C. The plates were then
washed and incubated with 100|J.1 of IX TMB substrate (Genei, Bangalore, India) and
absorbance was measured at 450nm using ELISA plate reader (Molecular Devices,
Sunnyvale, CA) after 5 minutes of color development at 25 C in the dark.
Direct Agglutination Test:
DAT was performed using serum samples following standard procedures, describe
elsewhere[22]. Briefly, serum at 1:400 dilution in PBS containing Ultrasore-G protein was
added in 96-well plates with V-shaped wells (8x12 Greiner, USA) and serially diluted from
1:400 to 1:25600. Fifty microliters freeze dried L.donovani antigen, prepared in ITMAntwerp,
Belgium as previously described, was dispensed to each well. Following overnight
incubation at room temperature, agglutination was observed and a DAT titer > 1:1600 was
taken as positive for L.donovani infection .
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9. Claim
We claim-
Soluble leishmania antigen specific whole blood methods as a marker of
asymptomatic infection and disease status of human visceral leishmaniasis: said
methods comprising the steps of-
I. Methods of stimulation of whole blood with SLA and measurements of IFN-y in
its 24 hrs. supematants as-a marker of cell mediated immunity / asymptomatic
infection.
11. Methods of stimulation of whole blood with SLA and measurements of lL-10 in
its 24 hrs. supematants for disease status of human visceral leishmaniasis.
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